[RASMB] Dissociation constants for protein/DNA complexes

[Borries Demeler]

Hi Sarah,
here is a publication that did it very similarly, except using fluorescence 
detection and using genetic algorithm analysis for quantification of 
the free and complexed portions (also accounting for free fluorophore,
if that is something you have present):

Zhuo Y, Ilangovan U, Schirf V, Demeler B, Sousa R, Hinck AP, Lafer EM.
Dynamic interactions between clathrin and locally structured elements in
a disordered protein mediate clathrin lattice assembly.  J Mol Biol. 2010
Nov 26;404(2):274-90. doi: 10.1016/j.jmb.2010.09.044. Epub 2010 Sep 25.

http://www.ncbi.nlm.nih.gov/pubmed/20875424

Regards, -Borries

On Thu, Jan 15, 2015 at 02:20:52AM +0000, Sarah Kessans wrote:
> Hi all,
> 
> We are trying a new (?) method of determining dissociation constants for protein/DNA complexes using velocity sedimentation and I am looking for feedback regarding the method.
> 
> Our method of determining Kds uses fluorescently-labeled oligos in complex with unlabeled protein.  By scanning at 488 nm, we can determine the size distribution of both unbound DNA and the protein/DNA complex.  We then calculate the area under each of the (baseline resolved) distribution curves, normalize these values to 100% total, and use the ratios (along with our known initial concentrations) to calculate the molar concentrations of bound/unbound protein/DNA.  From these data, we are able to get Kds that correspond well to Kds calculated from other experiments.
> 
> We are looking to publish these results, and I was wondering if anyone knew of Kds calculated in this manner before (either in published literature or otherwise)?  Any suggestions/comments welcome, thanks!
> 
> Best,
> Sarah
> 
> ___________________________
> Sarah Kessans
> Post-doctoral Fellow
> Biomolecular Interaction Centre
> School of Biological Sciences
> University of Canterbury
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